1. Field of the Invention
The present invention relates to FGF-1 and mutants of FGF-1 affecting the signaling of cellular growth, differentiation, and angiogenesis.
2. References
Various publications are referred to in parentheses throughout this application. Each of these publications is incorporated by reference herein. Complete citations of scientific publications are set forth below, or in the text of the specification.    Assoian, R. K. (1997) Anchorage-dependent cell cycle progression. J Cell Biol, 136, 1-4.    Belford, D. A., Hendry, I. A. and Parish, C. R. (1992) Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding. Biochemistry, 31, 6498-6503.    Brooks, P., Clark, R. and Cheresh, D. (1994a) Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science, 264, 569-571.    Brooks, P., Montgomery, A., Rosenfeld, M., Reisfeld, R., Hu, T., Klier, G. and Cheresh, D. (1994b) Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79, 1157-1164.    Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkar, F. H. and Cheresh, D. A. (1995) Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest, 96,.1815-1822.    Brown, K. J., Hendry, I. A. and Parish, C. R. (1995) Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: implications for potentiation of growth factor action by heparin. J Cell Biochem, 58, 6-14.    Burgess, W. H., Shaheen, A. M., Ravera, M., Jaye, M., Donohue, P. J. and Winkles, J. A. (1990) Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue. J Cell Biol, 111, 2129-2138.    Comoglio, P. M., Boccaccio, C. and Trusolino, L. (2003) Interactions between growth factor receptors and adhesion molecules: breaking the rules. Curr Opin Cell Biol, 15, 565-571.    DiGabriele, A. D., Lax, I., Chen, D. I., Svahn, C. M., Jaye, M., Schlessinger, J. and Hendrickson, W. A. (1998) Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature, 393, 812-817.    Eliceiri, B. P. (2001) Integrin and growth factor receptor crosstalk. Circ Res, 89, 1104-1110.    Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M., Vamer, J. A. and Cheresh, D. A. (1995) Definition of two angiogenic pathways by distinct alpha v integrins. Science, 270, 1500-1502.    Friesel, R. E. and Maciag, T. (1995) Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. Faseb J, 9, 919-925.    Frisch, S. M. and Screaton, R. A. (2001) Anoikis mechanisms. Curr Opin Cell Biol, 13, 555-562.    Fromm, J. R., Hileman, R. E., Weiler, J. M. and Linhardt, R. J. (1997) Interaction of fibroblast growth factor-1 and related peptides with heparan sulfate and its oligosaccharides. Arch Biochem Biophys, 346, 252-262.    Goodsell, D. S. and Olson, A. J. (1990) Automated docking of substrates to proteins by simulated annealing. Proteins, 8, 195-202.    Hynes, R. O. (2002) Integrins: bidirectional, allosteric signaling machines. Cell, 110, 673-687.    Kaplow, J. M., Bellot, F., Crumley, G., Dionne, C. A. and Jaye, M. (1990) Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek. Biochem Biophys Res Commun, 172, 107-112.    Klint, P. and Claesson-Welsh, L. (1999) Signal transduction by fibroblast growth factor receptors. Front Biosci, 4, D165-177.    Kwabi-Addo, B., Ozen, M. & Ittmann, M. The role of fibroblast growth factors and their receptors in prostate cancer. Endocr Relat Cancer 11, 709-24 (2004).    LaVallee, T. M., Prudovsky, I. A., McMahon, G. A., Hu, X. and Maciag, T. (1998) Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the Src pathway correlates with migration. J Cell Biol, 141, 1647-1658.    Lishko, V. K., Kudryk, B., Yakubenko, V. P., Yee, V. C. and Ugarova, T. P. (2002) Regulated unmasking of the cryptic binding site for integrin alpha M beta 2 in the gamma C-domain of fibrinogen. Biochemistry, 41, 12942-12951.    Liu, J., Huang, C. and Zhan, X. (1999) Src is required for cell migration and shape changes induced by fibroblast growth factor 1. Oncogene, 18, 6700-6706.    Morris, G. M., Goodsell, D. S., Halliday, R. S., Fig Huey, R., Hart, W. E., Belew, R. K. and Olson, A. J. (1998) Automated docking using a Lamarckian genetic algorithm- and an empirical binding free energy function. J Comp. Chem., 19, 1639-1662.    Morris, G. M., Goodsell, D. S., Huey, R. and Olson, A. J. (1996) Distributed automated docking of flexible ligands to proteins: parallel applications of AutoDock 2.4. J Comput Aided Mol Des, 10, 293-304.    Pages, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S. and Pouyssegur, J. (1993) Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A, 90, 8319-8323.    Pellegrini, L., Burke, D. F., von Delft, F., Mulloy, B. and Blundell, T. L. (2000) Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature, 407, 1029-1034.    Plotnikov, A. N., Hubbard, S. R., Schlessinger, J. and Mohammadi, M. (2000) Crystal structures of two FGF-FGFR complexes reveal the determinants of ligandreceptor specificity. Cell, 101, 413-424.    Powers, C. J., McLeskey, S. W. and Wellstein, A. (2000) Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer, 7, 165-197.    Prater, C. A., Plotkin, J., Jaye, D. and Frazier, W. A. (1991) The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J. Cell Biol., 112, 1031-1040.    Presta, M., Dell'Era, P., Mitola, S., Moroni, E., Ronca, R. and Rusnati, M. (2005) Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev, 16, 159-178.    Rusnati, M., Tanghetti, E., Dell'Era, P., Gualandris, A. and Presta, M. (1997) alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells. Molecular Biology of the Cell., 8, 2449-2461.    Sahni, A. and Francis, C. W. (2004) Stimulation of endothelial cell proliferation by FGF-2 in the presence of fibrinogen requires alphavbeta3. Blood, 104, 3635-3641.    Saphire, E. O., Parren, P. W., Pantophlet, R., Zwick, M. B., Morris, G. M., Rudd, P. M., Dwek, R. A., Stanfield, R. L., Burton, D. R. and Wilson, I. A. (2001) Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science, 293, 1155-1159.    Schlessinger, J. (2000) Cell signaling by receptor tyrosine kinases. Cell, 103, 211-225. Schwartz, M. A. and Assoian, R. K. (2001) Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways. J Cell Sci, 114, 2553-2560.    Schwartz, M. A. and Ginsberg, M. H. (2002) Networks and crosstalk: integrin signalling spreads. Nat Cell Biol, 4, E65-68.    Shimaoka, M. and Springer, T. A. (2003) Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discov, 2, 703-716.    Takagi, J., Erickson, H. P. and Springer, T. A. (2001) C-terminal opening mimics ‘inside-out’ activation of integrin alpha5beta1. Nat Struct Biol, 8, 412-416.    Tanghetti, E., Ria, R., Dell'Era, P., Urbinati, C., Rusnati, M., Ennas, M. G. and Presta, M. (2002) Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts. Oncogene, 21, 3889-3897.    Thornton, S. C., Mueller, S. N. and Levine, E. M. (1983) Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science, 222, 623-625.    Ullrich, A. and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell, 61, 203-212.    Wang, W. and Malcolm, B. A. (1999) Two-Stage PCR Protocol Allowing Introduction of Multiple Mutations, Deletions and Insertions Using QuikChange ™ Site-Directed Mutagenesis. BioTechniques, 26, 680-682.    Yokoyama, K., Erickson, H. P., Ikeda, Y. and Takada, Y. (2000) Identification of amino acid sequences in fibrinogen y-chain and tenascin C C-terminal domains critical for binding to integrin αvβ3. J. Biol. Chem., 275, 16891-16898.    Yokoyama, K., Zhang, X. P., Medved, L. and Takada, Y. (1999) Specific binding of integrin αVβ3 to the fibrinogen γ and αE chain C-terminal domains. Biochemistry, 38, 5872-5877.    Zhu, H., Anchin, J., Ramnarayan, K., Zheng, J., Kawai, T., Mong, S. and Wolff, M. E. (1997) Analysis of high-affinity binding determinants in the receptor binding epitope of basic fibroblast growth factor. Protein Eng, 10, 417-421.
3. Description of Related Art
Fibroblast growth factors (FGFs) constitute a family of heparin-binding polypeptides involved in the regulation of biological responses such as growth, differentiation, and angiogenesis. They are also implicated in inflammation, excess wound healing, and resistance of tumor cells to chemotherapeutic agents (chemoresistance).
The FGF family currently consists of 24 members, with FGF-1 (acidic FGF) and FGF-2 (basic FGF) the most extensively studied. The biological effects of FGFs are mediated by four structurally related receptor tyrosine kinases, denoted FGFR1, FGFR2, FGFR3, and FGFR4. The binding of FGF to its receptor results in receptor dimerization and subsequent autophosphorylation on specific tyrosine residues within the intracellular domain (Klint and Claesson-Welsh, 1999; Powers et al., 2000; Presta et al., 2005; Ullrich and-Schlessinger, 1990)
Integrins are a family of cell adhesion receptors that recognize extracellular matrix ligands and cell surface ligands (Hynes, 2002). Integrins are transmembrane α-β heterodimers, and at least 18 α and β subunits are known (Shimaoka and Springer, 2003). Integrins transduce signals to the cell upon ligand binding, and their functions are in turn regulated by the signals from within the cell (Hynes, 2002). Ligation of integrins triggers a large variety of signal transduction events that serve to modulate cell behavior including proliferation, survival/apoptosis, shape, polarity, motility, gene expression, and differentiation. Integrin-stimulated pathways are very similar to those triggered by growth factor receptors and are intimately coupled with them. It has been proposed that many cellular responses to soluble growth factors, such as epidermal growth factor, platelet-derived growth factor, and thrombin, are dependent upon the cell's adherence to extracellular matrix ligands via integrins. Integrins lie at the basis of such anchorage-dependent cell survival and proliferation (Assoian, 1997; Frisch and Screaton, 2001; Schwartz and Assoian, 2001).
It has been proposed that FGF-2-induced angiogenesis requires integrin signaling from the extracellular matrix (crosstalk between integrins and FGF receptors). Indeed antibody against integrin αvβ3 blocks FGF-2-induced angiogenesis (Brooks et al., 1994a; Brooks et al., 1994b). It has been reported that FGF-2 enhances αvβ3 expression during angiogenesis (Brooks et al., 1994a). Antibody or cyclic peptide antagonist of αvβ3 inhibits this αvβ3 upregulation (Brooks et al., 1994a; Brooks et al., 1995; Friedlander et al., 1995). It has been shown that integrin and growth factors are colocalized under certain condition. For example coimmunoprecipitation studies revealed direct biochemical interaction between αvβ3 and FGFR1 in the presence of both FGF-2 and fibrinogen (Sahni and Francis, 2004). These findings suggest integrin and FGFR are colocalized on the membrane in the presence of FGF-2. It has not been established how integrins and FGFR crosstalk in FGF-2 signaling.
It has been reported that substrate-bound FGF-2 promotes endothelial cell adhesion by interacting with integrin αvβ3 (Rusnati et al., 1997) and induces endothelial cell proliferation, motility, and the recruitment of FGFR1 in cell substrate contact (Tanghetti et al., 2002). Anti-αvβ3 antibodies block cell proliferation on immobilized FGF-2, but deletion of the tyrosine kinase portion of FGFR blocks cell proliferation induced by inmmobilized FGF-2. Thus it has been proposed that αvβ3 is required but not sufficient to transduce mitogenic signals of FGF-2 (Tanghetti et al., 2002). It is unclear how integrins interact with FGF-2 or whether this interaction is biologically relevant since heat-denatured FGF-2 still supports integrin binding (Tanghetti et al., 2002).